My laboratory is engaged in the study of gene expression in animal cells and animal viruses. The long term goal is to identify a protein chain-termination mutation in a structural gene, and then a revertant caused by a suppressor tRNA. Cells containing suppressor tRNAs would be of great value for the genetic analysis of animal viruses. Most of our effort is devoted to characterizing mutations in the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) in human cells. We have selected human HeLa and lymphoblast cells lacking HGPRTase activity by killing cells having HGPRTase activity with purine base analogs. We have selected HGPRTase revertants by blocking de novo synthesis with the folic acid antagonist methotrexate. To determine if the selected cells have altered HGPRTase, we have purified and characterized human HGPRTase from HeLa cells and red blood cells and prepared monospecific antibody against the enzyme. Using this antibody, we have devised methods which prepare apparently homogeneous HGPRTase protein from crude extracts of human cells in a single step (approximately 4000-fold purification). We are comparing purified mutant and wild-type HGPRTase proteins by peptide mapping utilizing high pressure liquid column chromatography. In addition to the immunological approach, we are characterizing HGPRTase mutants by two dimensional polyacrylamide gel electrophoresis of crude cell extracts. In this procedure, proteins are separated first in one dimension by isoelectric focusing, and then in a second dimension by sodium dodecyl sulfate gel electrophoresis. Although HGPRTase represents only 0.02% of the soluble protein in a HeLa cell extract, we have identified the spot corresponding to wild-type and mutant proteins in two-dimensional gels. BIBLIOGRAPHIC REFERENCE: G.S. Ghangas and G. Milman (1975). Radioimmune determination of hypoxanthine phosphoribosyltransferase cross-reacting material in erythrocytes of Lesch-Nyhan patients. Proc. Nat. Acad. Sci. 72: 4147-4150.